M.-Pilar Marco

Multifunctional nanoparticles--properties and prospects for their use in human medicine.

A major aim of medicine has long been the early and accurate diagnosis of clinical conditions, providing an efficient treatment without secondary effects. With the emergence of nanotechnology, the achievement of this goal seems closer than ever. To this end, the development of novel materials and devices operating at the nanoscale range, such as nanoparticles, provides new and powerful tools for imaging, diagnosis and therapy. This review focuses on the significant improvements in performance that nanoparticles offer compared with existing technologies relevant to medicine. Specifically, we address the design of multifunctional nanoparticles as an alternative system for drug and gene delivery, which has great potential for therapy in areas, such as cancer and neuropathologies. Moreover, we discuss the controversy generated by the possible toxic health effects of nanoparticles.

A label-free and portable multichannel surface plasmon resonance immunosensor for on site analysis of antibiotics in milk samples

Techniques for immunosensing like surface plasmon resonance (SPR) may respond to the need for rapid screening methods to improve food safety. This paper describes the development of a novel portable six channel SPR biosensor based on the plasmon of gold diffraction grating surface for simultaneous multianalyte antibiotic detection in milk samples. Representative congeners from three important antibiotic families (FQs: fluoroquinolones, SAs: sulfonamides and CAP: phenicols) were chosen for this study. The chips are covalently biofunctionalized with haptenized proteins by means of a previously formed mixed self assembled monolayer (m-SAM) prepared using two types of mercapto alkyl reagents containing polyethyleneglycol (PEG) units. The samples or standards are mixed with specific polyclonal antibodies and injected into the sensor device. The detectability accomplished is very good (i.e. in buffer, enrofloxacin, 0.30 μg L(-1); sulfapyridine, 0.29 μg L(-1); and chloramphenicol, 0.26 μg L(-1)) and whole milk samples can be analyzed directly without clean-up steps, by just diluting the sample five times with water to remove non-specific interferences caused by the matrix. Although the detectability of CAP regarding the MRPL (minimum required performance limit) is slightly compromised by the dilution, the detectability accomplished by FQs and SAs was far below the maximum residue levels (MRLs) established by the European Union.

An impedimetric immunosensor based on interdigitated microelectrodes (IDμE) for the determination of atrazine residues in food samples

A novel impedimetric immunosensor for atrazine detection has been developed. The immunosensor is based on an array of interdigitated micro-electrodes (IDmicroE) and immunoreagents specifically developed to detect this pesticide. Immunochemical determination of atrazine is possible without the use of any label. An atrazine-haptenized protein was covalently immobilized on the surface of the interdigitated mu-electrodes area (interdigits space) previously activated with (3-glycidoxypropyl)trimethoxysilane. Before, the gold electrodes were blocked using N-acetylcysteamine to prevent non-specific adsorptions. All biofunctionalization steps were characterized by chemical affinity methods and impedance spectroscopy. Immunosensors measures are made by exposing the sensor to solutions containing a mixture of the analyte and the specific antibody. With this configuration, the immunosensor detects atrazine with a limit of detection of 0.04 microg L(-1) without the use of any label. The potential of the immunosensor to analyze pesticide residues in complex sample matrices, such as red wine, has been evaluated. The results shown that after solid-phase extraction atrazine can be determined in this type of sample with a limit of detection of 0.19 microg L(-1), far below the Maximum Residue Level (MRL) established by EC for residues of this herbicide in wine.

Immunochemical Assays for Direct Sulfonamide Antibiotic Detection In Milk and Hair Samples Using Antibody Derivatized Magnetic Nanoparticles

Two direct enzyme-linked immunosorbent assays (ELISAs) have been developed for detection of sulfonamide antibiotic residues in milk samples. One of them is using magnetic nanoparticles (MNP) for target capture/enrichment (Ab-MNP-ELISA), and the second is performed using microtiter plates. Selective polyclonal antibodies, raised against 5-[6-(4-amino-benzenesulfonylamino)-pyridin-3-yl]-2-methyl-pentanoic acid (SA1), used in combination with an enzyme tracer prepared with the same hapten, has allowed us to reach a limit of detection (LOD) lower than 0.5 microg L(-1) for both ELISA formats. Sulfapyridine, sulfamethoxypyridazine, sulfathiazole, and sulfachloropyridazine are detected below the maximum residue limits established by the European Union for these antibiotics in milk (100 microg L(-1)). Matrix effects and accuracy studies performed with full-cream milk and hair extracts indicated a lack of interference from these sample matrices and very good recovery values, especially when using the Ab-MNP format. Milk samples and hair extracts can be measured without any previous treatment. The results demonstrate the high potential of these methods as screening tools for food safety and inspection controls.

A multianalyte ELISA for immunochemical screening of sulfonamide, fluoroquinolone and ß-lactam antibiotics in milk samples using class-selective bioreceptors

AbstractA multianalyte ELISA has been developed for the simultaneous determination of the most frequently used antibiotic families in the veterinary field following the typical planar microarray configuration, where the identity of the target analyte is encoded by its location in the detection platform (Master et al. in Drug Discovery Today 11:1007–1011, 2006). To accomplish this aim, two individual enzyme-linked immunosorbent assays for sulfonamide and fluoroquinolone antibiotics and an enzyme-linked receptor assay for ß-lactam antibiotics have been combined. The strategy uses microplates coated with the corresponding haptenized proteins in specific sections of the microplate. The samples are mixed with a cocktail containing the bioreagents, and distributed in the wells of the microplate. Identification of the antibiotic present in a particular sample is consequently accomplished by detecting a positive response on the corresponding microplate section. Since the bioreceptors used show a wide recognition of the congeners of each antibiotic family, the multianalyte method is able to detect more than 25 different antibiotics from the three most important antibiotic families. The detectability reached in full-fat milk samples is below the European maximum residue limits. The accuracy and reliability of this multiplexed bioanalytical method have been demonstrated by analyzing blind spiked samples. FigureSeveral milk samples can be screening for more than 25 antibiotic residues of β-lactam, fluoroquinolone or sulfonamide families in just two hours in a single microplate ELISA. Milk samples are mixed with a cocktail of bioreceptors and added to the microplate. The assay provides a specific response for each antibiotic family on a different microplate section.

Integrated disposable electrochemical immunosensors for the simultaneous determination of sulfonamide and tetracycline antibiotics residues in milk.

The design, preparation and analytical performance of a novel integrated amperometric immunosensor based on the immobilization of selective capture antibodies on the surface of Protein G-modified screen-printed dual carbon electrodes (SPdCEs) for the multiplexed determination of sulfonamide and tetracycline antibiotics residues in milk is reported in this work. Protein G was covalently immobilized onto a 4-aminobenzoic acid (4-ABA) film grafted on the disposable electrode, and a direct competitive immunoassay using horseradish peroxidase (HRP)-labeled tracers was performed. The amperometric responses measured at -0.2 V vs. the silver pseudo-reference electrode of the SPdCE upon the addition of H2O2 in the presence of hydroquinone (HQ) as mediator were used to monitor the extent of the immunoreactions. The developed methodology showed very low limits of detection (in the low ppb level) for sulfonamide and tetracycline antibiotics tested in untreated milk samples, and a good selectivity against other antibiotic residues frequently detected in milk and dairy products. The usefulness of the dual immunosensor was demonstrated by analyzing spiked milk samples as well as a reference milk containing a certified oxytetracycline (OTC) content. Good recoveries were attained in an analysis time of 30 min.

Impedimetric immunosensor based on a polypyrrole-antibiotic model film for the label-free picomolar detection of ciprofloxacin.

This paper describes the construction of an impedimetric immunosensor for the label-free detection of ciprofloxacin, an antibiotic belonging to synthetic fluoroquinolones. A poly(pyrrole-N-hydroxysuccinimide) film was electrogenerated onto electrodes and then used for the reagentless covalent binding of a fluoroquinolone model bearing an amino group. The resulting electrodes were utilized to immobilize a layer of anticiprofloxacin antibody onto the polymer surface by immunoreaction. In presence of ciprofloxacin, the antibody was displaced in solution inducing marked changes in the impedance of the sensor electrodes. These phenomena were detected and characterized by electrochemical impedance spectroscopy allowing the selective detection of extremely low ciprofloxacin concentration, namely, 1 x 10(-12) g mL(-1) or 3 pmol L(-1). Sensors exposed to ciprofloxacin showed a decrease in the sum of the interfacial resistances with the increase in ciprofloxacin concentration from 1 x 10(-12) to 1 x 10(-6) g mL(-1).

Portable Surface Plasmon Resonance Immunosensor for the Detection of Fluoroquinolone Antibiotic Residues in Milk

An inexpensive and portable surface plasmon resonance (SPR) sensor, SPReeta Evaluation Kit SPR3, has been used to develop a biosensor for the determination of fluoroquinolone antibiotics (FQs) and to demonstrate its performance analyzing FQ residues in milk samples. The SPReeta three-channel gold chips were activated with a mixed self-assembled monolayer (m-SAM) and functionalized with a FQ haptenized protein. Binding of the antibody produced a concentration-dependent increase of the SPR signal as a result of the change in the refraction index. Similarly, the presence of the FQ produced a dose-dependent decrease of the response, which allowed a good limit of detection (LOD) to be obtained (1.0 ± 0.4 μg L(-1) for enrofloxacin in buffer). The response was reproducible in all three channels, on different injections and days, and also between chips. Milk samples could be analyzed after a simple sample treatment involving fat removal by centrifugation and dilution with water. Under these conditions calibration curves were obtained showing that FQ residues can be analyzed in milk samples with an IC(50) value of 26.4 ± 7.2 μg L(-1) and a LOD of 2.0 ± 0.2 μg L(-1) (for enrofloxacin), far below the European Union regulations for this antibiotic family in this matrix. Finally, the paper also demonstrates that the biosensor is able to selectively detect the presence of FQs in milk samples, even in the presence of other antibiotics. Enrofloxacin, ciprofloxacin, and norfloxacin residues were detected in blind samples supplied by Nestlé Co.

Molecular Modeling Assisted Hapten Design To Produce Broad Selectivity Antibodies for Fluoroquinolone Antibiotics

Antibodies with a wide recognition profile of fluoroquinolone antibiotics have been produced based on chemical criteria, theoretical studies, and molecular modeling assisted hapten design. The immunizing hapten preserves the most important and characteristic epitopes of this antibiotic family. The studies have taken into consideration the zwitterionic character of most of the fluoroquinolones and the relative concentration of the different species in equilibrium at physiologic pH. The hapten is prepared in the form of a stable prehapten through a 5 step synthetic pathway. Immediately before conjugation, the immunizing hapten is obtained by removing the diphenylmethane protecting group. The specificity of the antibodies obtained is directed toward the common area defined by the fluorine atom at position 6 and the β-ketoacid moiety. The ELISA developed is able to recognize with very good detectability important fluoroquinolones used in the veterinary field such as ciprofloxacin (CPFX, IC(50), 0.35 μg L(-1)), enrofloxacin (ERFX, IC(50), 0.65 μg L(-1)), danofloxacin (DNFX, IC(50), 7.31 μg L(-1)), difloxacin (DFX, IC(50), 0.91 μg L(-1)), sarafloxacin (SRFX, IC(50), 0.96 μg L(-1)), norfloxacin (NRFX, IC(50), 0.78 μg L(-1)), ofloxacin (OFX, IC(50), 1.84 μg L(-1)), flumequine (Flume, IC(50), 3.91 μ gL(-1)), marbofloxacin (MBFX, IC(50), 4.30 μ gL(-1)), and oxolinic acid (OXO, IC(50), 23.53 μg L(-1)). The results presented here demonstrate that the antibody affinity is strongly affected by the presence of divalent cations, owing to their complexation with the fluoroquinolone molecules. Moreover, the outcome from the effect of the pH on the immunochemical assays suggests that the selectivity could be modulated with the pH due to the zwitterionic character of the fluoroquinolones and as a function of their different pK(a) values.

Waveguide interrogated optical immunosensor (WIOS) for detection of sulfonamide antibiotics in milk.

An immunosensor was developed for the detection of sulfonamide antibiotics in milk. Detection relied on a competitive immunoassay format, using immunoreagents previously developed for the generic detection of sulfonamide antibiotics and evaluated by enzyme-linked immunosorbent assay. The immunoassay was implemented onto a microsystem platform, the wavelength interrogated optical sensing device, which uses the evanescent field to probe changes at the interface of a waveguiding layer, and thus allows sensitive detection of biomolecule adsorption. The immunoreagents were immobilized onto the surface of the waveguide chip, and a fluidic cell allowed flowing analyte and detection solutions above the surface. Sulfapyridine was used as reference sulfonamide and detected with the immunosensor in buffer and in milk with a limit of detection (IC(90)) of 0.2+/-0.1 microg L(-1) and 0.5+/-0.1 microg L(-1), respectively. The analysis time was below 30 min, including regeneration of the sensing surface, with minimum sample preparation required. The reproducibility of the detection was better than 10%. A blind assay allowed identifying milk samples that were contaminated with different sulfonamide antibiotics at or above the maximum residue limits established by the European Union for these compounds (100 microg L(-1)). Thus, the developed immunosensor presents great potential as a generic sensing device for the fast and early detection of food contaminants on the field by non-skilled users.